The present study was carried out to determine if sensitization of Gram-negative bacteria to the polyanionic antibiotic daptomycin by cationic molecules can be explained on the basis of decreased cell surface charge in order to better understand intrinsic resistance. Turbidimetric assessments of batch cultural growth kinetics revealed the outer membrane permeabilizer polymyxin B nonapeptide sensitized Pseudomonas aeruginosa and Escherichia coli to the hydrophobic probe novobiocin, whereas little or no sensitization was observed for two surface hydrophobicity variants of Pasteurella multocida. Polymyxin B nonapeptide and daptomycin synergistically inhibited growth of P. aeruginosa only. A hydrocarbon adherence assay revealed permeabilizing concentrations of polymyxin B nonapeptide increased cell surface hydrophobicity of P. aeruginosa and the hydrophobic P. multocida variant, while E. coli and the hydrophilic P. multocida variant remained unaffected. Measurement of cellular electrophoretic mobility showed polymyxin B nonapeptide permeabilization of P. aeruginosa to daptomycin occurred concomitantly with a significant decrease in cell surface charge, while no such sensitization occurred in organisms which failed to undergo polymyxin B nonapeptide-induced surface charge decreases. These data suggest that sensitization of Gram-negative bacteria to polyanionic lipopeptides by growth in the presence of polycationic outer membrane permeabilizers such as polymyxin B nonapeptide is dependent on diminution of overall cell surface charge and polarity, thereby allowing outer cell envelope permeation.